The shikimate pathway constitutes an enormously significant route of metabolic flow in higher plants. Not only does it supply three of the amino acid residues used for protein assembly, but it supplies precursors for secondary metabolites in differentiated cells (e.g., lignin) which represents a huge fraction of metabolic output. The prime enzymatic link between aromatic amino-acid biosynthesis and secondars metabolism is phenylalanine ammonia lyase (PAL). We propose to study this metabolic interface via detailed enzyme studies comparing wild type and mutant cells of a true diploid species of tobacco, Nicotiana sylvestris. The enzymes that form arogenate (prehenate aminotransferase) or utilize argonate (arogoenate dehydratase and arogenate dehydrogenase) will be studied in relationship to PAL regulation. Assays will be carried out in both organismal tissues and cultured cells. Analogue-resistant mutants will be isolated in order to perturb biosynthesis and/or secondary metabolism in cultured cells (haploid and diploid). Temperature-sensitive mutants will also be isolated. All mutants will be regenerated to the organismal stage in order to relate gene-enzyme relationships to a developmental fram of reference. The effect of growth in the presence of PAL inhibitors and inducers (such as AOPP) will be investigated. N.sylvestris will be examined for the presence of isozymic forms of PAL (both chloroplast and cytoplasmic forms). Enzyme proteins detected will be purified to homgeneity and characterized. Any isozymes will be compared by amino acid sequencing and/or immunological comparisons. A molecular genetic approach will be started by isolation of mRNA for PAL. cDNA will be prepared for in situ hybridization onto chromosomes metaphase to describe the chromosomal location of the PAL gene. Structure of cDNA will be determined by restriction mapping.